Piperazides of substituted phenylalanine derivatives as thrombin inhibitors

ABSTRACT

The present invention relates to D,L-, L- and D-phenylalanine piperazides of formula (I) defined in claim 1 that inhibit blood coagulation, and thrombin and/or trypsin, respectively. The compounds are extraordinarily absorbable after oral, intraduodenal and in particular rectal administration and show only a low toxicity.

This application is the national stage of PCT/CH94/00026, filed Feb. 9,1994.

The present invention relates to new proteinase inhibitors which containphenylalanine as the core structure, wherein the aromatic residue inmeta position carries a basic group and the a-amino group issulfonylated with various residues. The C-terminal introduction ofdifferent, N-substituted piperazine residues led to the discovery ofhighly efficient inhibitors showing improved bioavailability.

Proteinase inhibitors are potential drugs which can be used to controlphysiological processes induced and maintained by proteinases. For manyendogenous and naturally occurring inhibitors, respectively, it has beenshown that they can influence the activity of proteinases in vivo andalleviate hyperproteolytic states [see H orl, W. H. In: Design of EnzymeInhibitors as Drugs, p. 573-581, (Sandler, M. and Smith, H. J., Eds.)Oxford, New York, Tokyo: Oxford University Press, 1989]. However, thetherapeutic application of such inhibitors of relatively high molecularweight is limited due to their particular protein structure. As theseinhibitors are not absorbed in the intestine upon oral administration onthe one hand and exert an antigenic activity on the other hand, it wasof great interest to search for synthetic enzyme inhibitors of lowmolecular weight.

The four classes of enzymes which are responsible forproteinase-dependent processes comprise the serine, thiol, metallo, andaspartate proteinases. Serine proteinases are proteolytic enzymes whichpossess a reactive serine residue in the active center. Enzymes which,such as trypsin, split off C-terminal peptide bonds of the basic aminoacids arginine and lysine, belong to the trypsin family of the serineproteinases. In this group, those enzymes which induce coagulation andfibrinolysis in the blood, which release kinin and produce thecomplement activation or those which themselves are components of thementioned enzyme systems, are of particular physiological significance.

Blood coagulation is triggered by zymogen activation via two differentpathways. The first, intrinsic pathway leads to blood coagulation via achain of reactions mediated by blood constituents. The second, extrinsicpathway leads to coagulation via a shorter chain of reactions based onan interaction between blood and tissue constituents. Both ways producethe activation of the zymogen factor X into the serine proteinase factorX_(a) which itself catalyzes the activation of prothrombin into thefibrinogen-coagulating serine proteinase, thrombin. Being a commonproduct of the intrinsic as well as of the extrinsic activation pathway,factor X_(a) was initially considered to be the preferential targetenzyme for inhibitory intervention in the blood coagulation process(Tidwell, R. R. et al., Thromb. Res. 19, 339-349, 1980). However, it hasbeen recently demonstrated that synthetic inhibitors of factor X_(a) invitro and in vivo are not coagulation-inhibiting (St urzebecher, J. etal., Thromb. Res. 54, 245-252, 1989) and antithrombotically efficient(Hauptmann, J. et al., Thromb. Haemostas. 63, 220-223, 1990). Therefore,the development of anticoagulant inhibitors is focused on the discoveryof thrombin inhibitors.

For the development of synthetic inhibitors for thrombin, benzamidinederivatives have been extensively investigated (St urzebecher, J. etal., Acta Biol. Med. Germ. 35, 1665-1676, 1976). Among them, amino acidderivatives containing a benzamidine moiety and a para-oriented amidinogroup proved to be favorable core structures for the development ofeffective inhibitors. Until now, the most efficient thrombin inhibitorof the benzamidine type described is the amino acid derivativeNα-(2-naphthylsulfonyl)-glycyl-4-amidinophenylalanine piperidide (NAPAP)(K_(i) =6×10 ⁻⁹ mol/l ) (St urzebecher, J. et al., Thromb. Res. 29,635-642, 1983).

Other types of inhibitors which also effectively inhibit thrombin areknown: a first group is comprised of the peptidyl-arginine-chloromethylketones, e.g. H-D-Phe-Pro -Arg-CH₂ Cl (Kettner, C. et al., Thromb. Res.14, 969-973, 1979). A second group is comprised of the peptidylargininealdehydes, e.g. Boc-D-Phe-Pro-Arg-H and H-D-Phe-Pro-Arg-H (Bajusz, S.,Int. J. Peptide Protein Res. 12, 217-221, 1978).

However, these inhibitors which inhibit trypsin and thrombin with acomparable affinity are not easily synthesized, are unstable and maycause undesired side reactions due to their high reactive capacity.Thrombin, and trypsin as well, are inhibited in a time-dependentreaction by the boronic acid derivative Boc-D-Phe-Pro-Boro-Arg-C₁₀ H₁₆(see European Patent Application No. 0 293 881). Conversely, theselective thrombin inhibitor (2R,4R)-4-methyl-1-[Nα-(3-methyl-1,2,3,5-tetrahydro-8-quinolinesulfonyl)-L-arginine]-2-pipecoline-carboxylicacid has practically no trypsin-inhibiting activity (Kikumoto, R. etal., Biochemistry 23, 85-90, 1984).

All the benzamidine derivatives investigated until now possesspharmacodynamic and pharmacokinetic properties which make themunfavorable for a therapeutic application. Upon oral application theyare not absorbed in the intestine, they are quickly eliminated from thecirculation and their toxicity is relatively high. This applies to theamides of the N-α-arylsulfonylated (Markwardt, F. et al., Thromb. Res.17, 425-431, 1980) as well as to amides of theN-α-aryl-sulfonylaminoacylated 4-amidinophenylalanine (see PatentApplication No. DD-A-235 866). The responsibility for the inadequatepharmacological properties is attributable to the strongly basic amidinofunction (Kaiser, B. et al., Pharmazie 42, 119-121, 1987). Experimentsaimed at replacing the strongly basic amidino function in highlyeffective inhibitors by less basic groups first failed as they resultedin a significant loss in efficacy (St urzebecher, J. et al., Pharmazie43, 782-783, 1988). Also, the introduction of a carboxyl group in theinhibitor to reduce the basicity of the amidino function led to adecrease in the inhibitory activity. Thus, derivatives of the4-amidinophenylalanine which possess a C-terminal amino acid with a freecarboxyl group are fully ineffective as inhibitors (Wagner, G. et al.,Pharmazie 39, 16-18, 1984; Vieweg, H. et al., Pharmazie 39, 82-86,1984).

The modification of NAPAP by the introduction of a substituent at theα-nitrogen led to a slight increase in the antithrombin activity (seeEuropean Patent Application No. FR-A-2 593 812), neither improved thepharmacological properties (Cadroy, Y. et al., Thromb. Haemostas. 58,764-767, 1987).

Starting from Nα-substituted 3-amidinophenylalanine, the development ofselective thrombin inhibitors was continued; it has been found thatamides of the Nα-2-naphthylsulfonylated 3-amidinophenylalanine type witha carboxylic group in the amide moiety as well as those derivatives inwhich the amidino function was replaced by another basic group showedimproved pharmacological properties. In particular, a certain absorptionhas been found for the first time in benzamidine derivatives after oralapplication (PCT/CH 9100235).

This class of substances was then further developed. It has been found,for example, that by introducing new substituents at the N-4-atom ofN-α-sulfonylated 3-amidinophenylalanine piperazides, in particular byintroducing acyl- (--CO--X), sulfonyl (--SO₂ --Y),carbamoyl-(--CO--NR'R") or functionalized alkyl residues, wherein X, Yand R',R" in the favorable case represent methyl groups and thefunctionalized alkyl residue (C₁ -C₃) carries an OH group, theinhibitory efficacy against thrombin could be significantly improved andan extraordinary increase of the resorption capability could besurprisingly noted. This was observed in particular after rectal andintraduodenal application of the derivatives which were used either assalts or as free bases.

Not only the racemic mixtures, but also the pure optical antipodes wererepresented.Nα-(2-Naphthylsulfonyl)-3-amidino-(L)-phenylalanine-4-acetylpiperazidefor example was synthesized within this framework. It has been foundthat this compound is not only a potent thrombin inhibitor andefficiently influences the coagulation, but that it surprisinglypossesses improved pharmacokinetic properties. It is absorbed by theintestine in particular after rectal administration to rats and ispresent in the blood for a relatively long time period in a bloodcoagulation-inhibiting and antithrombotically efficient concentration.This also applies to compounds with other N-terminal protective groupswhich carry an N-substituted piperazine as the amide residue.

Besides effective and biologically available thrombin inhibitors,trypsin inhibitors which were highly effective in case of reduction inthrombin activity and which were also extensively absorbed after rectalapplication, were found in the presented class of substances amongderivatives carrying at the piperazide nitrogen e.g. a heteroaryl or anacyl residue (--CO--X), wherein X represents a straight or branchedalkyl (C₃ -C₁₀), an aralkyl or a cycloalkyl residue (C₃ -C₁₀). Theinhibition of trypsin activity by inhibitors in hyperproteolytic statesin the pancreas is of high therapeutic interest.

The present invention relates to new proteinase-inhibiting phenylalaninepiperazides of the general formula I ##STR1## which can be present asracemates or as L- or D-configurated compounds and wherein R¹ representsa basic group of formula ##STR2## R² represents an (un)substituted arylor heteroaryl residue e.g. phenyl, 4-methylphenyl, 2,4,6-trimethyl- or-triisopropylphenyl, 4-methoxy-2,3,6-trimethylphenyl,2,2-dimethyl-6-methoxy- or 2,2,5,7,8-pentamethylchromanyl,anthraquinonyl, 1- or 2-naphthyl, quinolyl- or isoquinolyl or a camphorresidue,

and

R³ represents an acyl residue of formula --COX wherein X means H,unbranched or branched, possibly substituted alkyl, preferentially lowalkyl, in particular methyl, (un)substituted aryl or cycloalkyl,preferentially C₃ -C₁₀,

an aralkyl residue in which the aromatic residue may be substituted withe.g. a halogen atom, an alkyl, alkoxy, hydroxy or nitro group,

a carboxamide residue of formula --CONR'R", a thiocarboxamide residue offormula --CSNR'R" or an ethylamide residue of formula --CH₂ --CONR'R" inwhich R'═R"═H; R'═R"═alkyl; R'═H, R"═alkyl; R'═H, R"═aryl, or R' and R"may form a cycloaliphatic or heterocycloaliphatic ring with the nitrogenatom,

an SO₂ --Y residue in which Y means (un)substituted alkyl,preferentially methyl, trifluoromethyl, trichloromethyl, (un)substitutedaryl or heteroaryl, e.g. phenyl, 4-methylphenyl, 2,4,6-trimethyl- or-triisopropylphenyl, 4-methoxy-2,3,6-trimethylphenyl,2,2-dimethyl-6-methoxy- or 2,2,5,7,8-pentamethylchromanyl,anthraquinonyl, naphthyl or quinolyl or O-aryl, preferentially phenyl,or -NR'R", in which R'and R"═H may be equal or not to low alkyl C₁ -C₃,

a cycloaliphatic ring with 5 to 8 C atoms which may be substituted witha hydroxyl or oxo group,

an (un)substituted heteroaryl residue, e.g. pyridyl or pyrimidyl, or aheterocycloaliphatic residue, e.g. N-methylpiperidyl,

a functionalized alkyl residue of formula --(CH₂)_(n) --X wherein thealkyl chain may be unbranched or branched, n=1 to 8 and the functionalresidue X represents

a hydroxyl group the H atom of which can be substituted with an alkyl,aralkyl, aryl, hydroxyalkyl or acyl group,

a halogen atom,

a tertiary amino group of formula --N(Alk)₂ wherein the alkyl groupshave 1 to 3 C atoms and the same denotation and, moreover, the nitrogenatom may belong to a cycloaliphatic ring with 5 to 7 ring parts to whichone or two further rings may be added,

an acylaminomalonate group of formula ##STR3## an ##STR4## group,wherein Ac generally means formyl or acetyl and Alk=low alkyl or a##STR5## group, and the salts thereof with mineral or organic acids.

Among the phenylalanine piperazides defined in the general claims, thecompounds wherein R¹ represents a basic group of formula (a)=amidino, R²represents a β-naphthyl, anthraquinone, 2,4,6-triisopropylphenyl and2,2,5,7,8-pentamethylchroman group, and R³ represents an acyl residue,in particular formyl and acetyl, a functionalized alkyl residue, e.g.2-hydroxyethyl, an SO₂ --Y residue, a carboxamide residue as well asheteroaryl residues such as 2-pyridyl or 2-pyrimidyl, are of particularimportance.

Compounds of general formula I wherein R¹ =amidino (a) are synthesizedaccording to the known methods described hereinafter.

(D,L)-3-Cyanophenylalanine alkylesters of general formula II areconverted in an adequate solvent with a sulfochloride of general formulaIII, wherein R² has the denotations described in general formula I, intothe racemic compounds of general formula IV, from which racemates of thesulfonylated cyanophenylalanines V are obtained by acidic or alkalinehydrolysis.

The L-configurated sulfonylated amino acids V can be obtained byenzymatic ester hydrolysis of the compounds IV with chymotrypsin in anacetonitrile/water mixture. The D-configurated, sulfonylated amino alkylcarboxylates IV obtained by this procedure are converted intoD-configurated sulfonylated amino carboxylic acids V by acidichydrolysis in a mixture of 1N HCl and acetic acid by heating underreflux.

The cyano compounds with piperazide structure VI, which may have aracemic, or L or D configuration, respectively, can be obtainedaccording to the usual coupling procedures from the correspondinglyconfigurated compounds V with a piperazine derivative of general formulaVII.

Moreover, the piperazides VI can be obtained by a principally knownmethod consisting in first protecting racemic, L- orD-3-cyanophenylalanine by the introduction of a Boc group at the aminofunction. The obtained carboxylic acid V, with Boc group instead of SO₂--R², is transformed by conversion with a piperazine derivative VII, ina corresponding Boc-protected compound VI from which the cyano compoundswith piperazide structure VI are obtained after acidic cleavage of theBoc group and conversion with a sulfochloride of general formula III.

Addition of H₂ S to the cyano function gives the thioamides VIII whichare converted into the thioimidate halides IX by conversion with analkyl halide. Moreover, the imidate halides X can be obtained from thecyano compounds VI in a known way.

To represent the target compounds of general formula I wherein R¹=amidino (a), having a racemic, or L or D configuration, respectively,and wherein R² and R³ have the denotations mentioned in the generalformula I and X=halogen, preferentially chlorine, the thioimidate saltsIX are converted into compounds of general formula I in an alcoholicsolution with ammonium acetate or the imidate salts X are converted intocompounds of general formula I in an alcoholic ammonia solution. Theamidine salts thereby obtained can be converted in an adequate way intofree bases.

The compounds of general formula I wherein R¹ =aminomethyl (b) areobtained from the cyano compounds VI by catalytic hydrogenation, e.g.Raney-Nickel/hydrogen in adequate solvents in the presence of ammonia.##STR6##

The biological activity of the compounds of the present invention wasdetermined in vitro as well as in vivo. For characterizing theinhibitory activity in vitro, the dissociation constants K_(i) for theinhibition of thrombin and the related enzymes trypsin, plasmin, factorX_(a), factor XII_(a), plasma kallikrein, glandular kallikrein and tPA,respectively, were calculated according to the formula ##EQU1## wherein[E] represents the concentration in free enzyme, [I] the concentrationin free inhibitor and [EI] the concentration in enzyme-inhibitor complex(Dixon, Biochem. J. 55, 170-173 [1953]). The smaller the K_(i) -valuefor a tested enzyme, the higher the affinity of the inhibitor for theenzyme and the smaller the quantity of inhibitor needed for theinhibition of the enzyme, e.g. thrombin.

Various coagulation tests were used in vitro to determine the efficacyof the inhibitors towards the thrombin-induced coagulation of itsnatural substrate fibrinogen. For that purpose, the thrombin time (TT),the activated partial thromboplastin time (aPTT) and the prothrombintime (PT, Quick value) were determined in human plasma.

The toxicity of the compounds of the present invention was evaluated bydetermination of the LD₅₀ (=dose that causes the death of 50% of thetest animals within an observation time of one week) in the mouse afterintravenous and peroral administration, respectively.

For the pharmacokinetic characterization, the plasma concentration ofselected derivatives was determined in rats after intravenous (i.v.),peroral (p.o.), intraduodenal (i.d.) and rectal application according tothe following three-step procedure:

1. A physiological NaCl solution of the substance to be tested wassubmitted to high pressure liquid chromatography (HPLC) in order todetermine its characteristic substance-specific retention time with thechosen test conditions.

2. The substance to be tested was diluted in vitro in rat plasma. Thissolution was also submitted to HPLC to see whether the characteristicpeak of the substance once again appeared at the substance-specificretention time.

3. The substance to be tested was dissolved in physiological NaClsolution and administered i.v., p.o., i.d. and rectally, respectively,to rats in doses of 1, 50 and 100 mg per kg body weight. Blood sampleswere taken at time intervals of 15 minutes, from which plasma sampleswere prepared by centrifugation; those samples were also submitted toHPLC to see whether the characteristic peak of the substance appearedagain at the substance-specific retention time.

To demonstrate the pharmacological efficacy, the substance to be testedwas dissolved in physiological NaCl solution and administered rectallyto rats in doses of 5 and 20 mg per kg body weight, respectively. Bloodsamples were taken at time intervals, from which plasma samples wereprepared by centrifugation and investigated in the coagulation test(thrombin time TT and activated partial thromboplastin time aPTT).

The antithrombotic activity of the compounds was determined in the raton the model of the stasis-induced thrombosis according to Wessler etal. (J. Appl. Physiol. 14, 943-946, 1959). The thrombus was induced byserum 30 min after application of the inhibitor and macroscopicallyevaluated after 10 further min.

The compounds of the present invention can be used as diagnostics ordrugs in adequate application forms either as their salts or free bases.

The invention is explained in detail in the four examples describedhereafter.

EXAMPLE 1 Nα-(2-naphthylsulfonyl)-3-amidino-(L)-phenylalanine-4-acetyl-piperazide

Nα-(2-naphthylsulfonyl)-3-cyano-(D,L)-phenylalaninemethyl-ester (formulaIV; Alk=--CH₃, R² =β-naphthyl)

24.1 g (0.1 mol) of 3-cyano-(D,L)-phenylalaninemethylester hydrochloridewas suspended in 200 ml of dioxane, 20.6 g (0.204 mol) ofN-methylmorpholine was added under stirring and a solution of 23.6 g(0.104 mol) of 2-naphthylsulfonylchloride in 200 ml of ethyl acetate wasadded dropwise. The mixture was stirred for 16 hours at roomtemperature, the precipitated N-methylmorpholine hydrochloride wasfiltered off and the solvent evaporated under reduced pressure. Theresidue was dissolved in 50 ml of methanol, 50 ml of diethylether wasadded and the mixture was allowed to crystallize. The formed precipitatewas filtered off, washed with diethylether and dried in a vacuumdesiccator (KOH/H₂ SO₄). Yield: 36 g (91.3%). Mp: 122°-123° C. TLC:R_(f) =0.65 (chloroform 40/methanol 4/acetic acid 1//v/v/v/)

Nα-(2-naphthylsulfonyl)-3-cyano-(L)-phenylalanine (formula V; R²=β-naphthyl)

17.4 g (0.044 mol) of Nα-(2-naphthylsulfonyl)-3-cyano-(D,L)phenylalaninemethylester was dissolved in 260 ml of acetonitrile, 130 ml of water,100 mg of chymotrypsin as well as 0.785 g of potassium chloride wasadded and the pH of the solution was adjusted to 6.8-7 with 2 N NaOH.After stirring for 24 hours at room temperature, another 50 mg ofchymotrypsin were added to the mixture after 5 and again after 10 hoursand the above mentioned pH of the solution was maintained by controlledaddition of a total of 11 ml of 2 N NaOH. After subsequent filtration,the acetonitrile was evaporated under reduced pressure and the aqueoussolution was extracted several times with ethyl acetate. Afteracidification with 1 N HCl the aqueous phase was again extracted withethyl acetate, the collected organic phases were washed with saturatedsodium chloride solution, dried over magnesium sulfate, and the solventwas evaporated under reduced pressure, whereupon the product began tocrystallize towards the end of the distillation. After interruption ofthe distillation, 30 ml of diethylether and 70 ml of hexane was added,the precipitate was filtered off, washed with a small amount ofdiethylether and dried. Yield: 7.2 g (85.8%). Mp: 192°-193° C. [α]_(D)²⁰ =+11.9° (C=3, in methanol) TLC: R_(f) =0.25 (chloroform 40/methanol4/acetic acid 1//v/v/v)

Nα-(2-naphthylsulfonyl)-3-cyano-(L)-phenylalanine-4-acetylpiperazide(formula VI; R² =β-naphthyl, R³ =--COCH₃)

1.59 g (13.2 mmol) of 1-acetylpiperazine was dissolved in 10 ml of THFand 20 ml of DMF; the solution was mixed with 1.28 g (7.9 mmol) of HOBtand 2.5 g (6.6 mmol) of Nα-(2-naphthyl-sulfonyl)-3-cyano-(L)-phenylalanine and cooled to 0° C. After addition of 1.5 g(7.3 mmol) of DCC, the solution was stirred for 2 hours at 0° C. andthen for 22 hours at room temperature. The precipitated dicyclohexylurea was filtered off and the solvent evaporated under reduced pressure.The residue was dissolved in chloroform and purified by columnchromatography over silica gel 60 with chloroform/methanol 93:7 as theeluent. 2.9 g (90%) of an amorphous product were obtained.

[α]_(D) ²⁰ =+46.3° (C=1, in methanol) TLC: R_(f) =0.36 (chloroform40/methanol 4/acetic acid 1//v/v/v)

Nα-(2-naphthylsulfonyl)-3-thiocarboxamido-(L)-phenylalanine4-acetylpiperazide (formula VIII; R² =β-naphthyl, R³ =--COCH ₃)

2.75 g (5.6 mmol) ofNα-(2-naphthylsulfonyl)-3-cyano-(L)-phenylalanine-4-acetylpiperazide wasdissolved in 25 ml of pyridine. Twenty drops of TEA were added to thesolution which was saturated by introducing H₂ S for 10 min. Thereaction mixture was allowed to stand at room temperature for 2 days,whereupon the solvent was evaporated and the residue was dissolved inethyl acetate and extracted with 1 N HCl The organic phase was washedwith saturated sodium chloride solution, dried over magnesium sulfateand the solvent was evaporated. 2.6 g (88%) of yellow, amorphous productwere obtained and further processed in this form.

Nα-(2-naphthylsulfonyl)-3-S-methyliminothiocarbonyl-(L)-phenylalanine-4-acetylpiperazide hydroiodide (formula IX; Alk=--CH₃,X=I, R² =β-naphthyl, R³ =--COCH₃)

2.6 g (4.96 mmol) of the previously described thioamide was dissolved in60 ml of acetone, 6 g (42.3 mmol) of methyliodide was added to thesolution and the reaction mixture was allowed to stand in the dark for20 hours at room temperature. Afterwards, the solvent was evaporated,the oily residue was triturated with isopropanol/diethylether, theobtained powder was filtered off, washed with diethylether and dried.Yield: 3.1 g (94%).

Nα-(2-naphthylsulfonyl)-3-amidino-(L)-phenylalanine-4-acetylpiperazidehydrochloride (formula I; X=Cl, R² =β-naphthyl, R³ =--COCH₃)

3.0 g (4.5 mmol) of methyl-thioimidate hydroiodide was dissolved in 100ml of methanol, 0.8 g (10.4 mmol) of ammonium acetate was added to thesolution and the test mixture was heated for 3 hours at 60° C. in awater bath. The solvent was then evaporated, the residue was dissolvedin warm isopropanol and the amidine hydroiodide was precipitated withethyl acetate, filtered off, washed with ethyl acetate and diethyletherand dried. For the conversion into the hydrochloride, the obtainedproduct was dissolved in methanol and the solution passed over astrongly basic ion exchanger (Amberlite IRA-410, loaded with Cl⁻). Thehydrochloride was precipitated from the concentrated methanolic solutionwith ethyl acetate/diethylether 1:1. Yield: 1.8 g (73.5%). [α]_(D) ²⁰=+61.8° (C=1 in methanol) TLC: R_(f) =0.2 (organic phase of ethylacetate 4/acetic acid water 2//v/v/v) Specific rotation of thecorresponding (D)-configurated compound: [α]_(D) ²⁰ =-62.2° (C=1, inmethanol)

EXAMPLE 2 Nα-(2-naphthylsulfonyl)-3-amidino-(L)-phenylalanine-4-(2hydroxyethyl) -piperazide

Nα-(2-naphthylsulfonyl)-3-cyano-(L)-phenylalanine-4-(2hydroxyethyl)-piperazide hydrochloride (formula VI; R² =β-naphthyl, R³ =--CH₂ CH₂ OH)

2.0 g of Nα-(2-naphthylsulfonyl)-3-cyano-(L)-phenylalanine was added to10 ml of thionyl chloride and the mixture was heated for 30 min. underreflux. After cooling, the obtained solution was treated with hexaneuntil a turbid solution was obtained. The crystallized acid chloride wasfiltered off after 1 hour, washed with hexane and vacuum-dried. 1.7 g(4.26 mmol) of this product was dissolved in 25 ml of THF and addeddropwise within 15 min. under stirring to a solution of 1.16 g (8.9mmol) of 1-(2-hydroxyethyl)-piperazine in 15 ml of THF. After 1 hour ofstirring, the precipitated 1-(2-hydroxyethyl)-piperazine hydrochloridewas filtered off and the solvent evaporated. The remaining residue wasdissolved in 15 ml of methanol, water was added until a turbid solutionwas obtained, the mixture was allowed to stand overnight, whereby thepiperazide separated itself as an oil. After removal of the supernatant,the oil was taken up in 100 ml of ethyl acetate, the ethyl acetatesolution was washed with saturated sodium chloride solution, dried overMgSO₄ and concentrated under vacuum. The resulting solution wasacidified with 2 N ethyl acetate/HCl whereupon 50 ml of diethyletherwere added. The formed precipitate was filtered off after 1 hour ofstanding, washed with diethylether and vacuum-dried. Yield: 1.65 g(73%). [α]_(D) ²⁰ =-5.4° (C=1, in methanol) TLC: R_(f) =0.43 (organicphase of ethyl acetate 4/acetic acid 1/water 2//v/v/v)

Nα-(2-naphthylsulfonyl)-3-methoxyiminocarbonyl-(L)-phenylalanine-4-(2-hydroxyethyl)-piperazidedihydrochloride (formula X; Alk=--CH₃, X=Cl, R² =β-naphthyl, R³ =--CH₂CH₂ OH)

1.4 g (2.65 mmol) of the previously described cyano compound wasdissolved in a mixture of 7.5 ml of abs. methanol and 10 ml of abs.dioxane, 5.2 g (0.143 mol) of dried HCl gas was introduced into thesolution under ice cooling and the mixture was kept for 3 days in therefrigerator. Afterwards, the mixture was poured into 150 ml ofdiethylether, the formed precipitate was worked up after removal of thesupernatant, suspended in 40 ml of abs. ethanol, the crystalline powderfiltered off, washed with ethanol, diethylether and dried. Yield : 1.44g (91%). TLC: R_(f) =0.15 (organic phase of ethyl acetate 4/acetic acid1/water 2//v/v /v) TLC: R_(f) =0.95 (chloroform 70 /methanol 42/aceticacid 0.5/ water 10//v/v/v)

Nα-(2-naphthylsulfonyl)-3-amidino-(L)-phenylalanine-4-(2-hydroxyethyl)-piperazide dihydrochloride (formula I; X =Cl, R² =β-naphthyl, R³ =--CH₂CH₂ OH)

1.3 g (2.18 mmol) of the previously described methylimidatedihydrochloride was suspended in 30 ml of methanol and ethanolic ammoniasolution was added under stirring until a pH of 8.7 was reached, wherebya clear solution was obtained. The mixture was heated for 3 hours at 60°C. in a water bath, the solvent was then evaporated under reducedpressure, the residue was dissolved in 15 ml of abs. ethanol and, afteraddition of 20 drops of 2N ethyl acetate/HCl, the amidinedihydrochloride was precipitated with ethyl acetate, filtered off,washed with ethyl acetate and diethylether and dried. Yield: 1.02 g(80.3%). [α]_(D) ²⁰ =+14.2° (C=1, in methanol) TLC: R_(f) =0.18 (organicphase of ethyl acetate 4/acetic acid 1/water 2//v/v/v) TLC: R_(f) =0.6(chloroform 70/methanol 42/acetic acid 0.5/ water 10//v/v/v)

Specific rotation of the corresponding (D)-configurated compound:[α]_(D) ²⁰ =15.0° (C=1, in methanol)

To obtain the free base, 0.5826 g (1 mmol) of amidine dihydrochloridewas dissolved in 20 ml of methanol, the equimolar quantity of 0.1N NaOH(20.00 ml) was added to the solution and the solvent was evaporatedunder reduced pressure. To remove the still present water traces, aco-distillation with toluene/isopropanol was performed several times.The base obtained in this way still contains NaCl. To eliminate theinorganic component, a mixture of 15 ml of abs. ethanol, 10 ml ofchloroform and 10 ml of diethylether was added, whereby after stirring,the base dissolved. Insoluble NaCl was filtered off and the solventevaporated under reduced pressure. The remaining residue solidified whentriturated with diethylether. Yield: 0.48 g (94%)

EXAMPLE 3Nα-(2-naphthylsulfonyl)-3-amidino-(L)-phenylalanine-4-methylsulfonylpiperazide

Nα-(2-naphthylsulfonyl)-3cyano-(L)-phenylalanine-4-methylsulfonylpiperazide(formula VI, R² =β-naphthyl, R³ =--SO₂ CH₃)

1.56 g (7.8 mmol) of 1-methylsulfonylpiperazine.HCl was suspended in 15ml of DMF, the suspension was mixed under stirring with 0.86 ml (7.8mmol) of NMM, 1.16 g (7.8 mmol) of HOBt, 2.7 g ofNα-(2-naphthylsulfonyl)-3-cyano-(L)-phenylalanine (7.1 mmol) and 70 mlof THF and cooled to 0° C. After addition of 1.61 g of DCC (7.8 mmol),the mixture was stirred for another 20 hours at room temperature.Afterwards, the precipitated dicyclohexyl urea was filtered off and thesolvent evaporated under reduced pressure. The residue was dissolved inchloroform and purified by column chromatography over silica gel 60 withchloroform as the eluent. 3.34 g (89%) of an amorphous product wereobtained.

[α]_(D) ²⁰ =+47.3° (C=1, in methanol) TLC: R_(f) =0.36 (chloroform40/methanol 4/acetic acid 1//v/v/v)

Nα-(2-naphthylsulfonyl)-3-thiocarboxamido-(L)-phenylalanine-4-methylsulfonylpiperazide(formula VIII; R² =β-naphthyl, R³ =SO₂ CH₃)

2.9 g (5.2 mmol) of the previously described cyano compound wasdissolved in 35 ml of pyridine, 15 drops of TEA was added and thesolution was saturated by 10-min. introduction of H₂ S. The mixture wasallowed to stand at room temperature for 2 days. The solvent was thenevaporated under reduced pressure and the residue dissolved in ethylacetate, whereby the thioamide progressively crystallized out. Theprecipitate was filtered off, washed with ethyl acetate and dried.Yield: 2.98 g (96%).

Nα-(2-naphthylsulfonyl)-3-S-methyliminothiocarbonyl-(L)-phenylalanine-4-methylsulfonylpiperazide(formula IX; Alk =CH₃, X=I, R² =β-naphthyl, R³ =--SO₂ CH₃)

2.95 g (5.26 mmol) of the described thioamide was dissolved in 4 ml ofDMF under heating, to the solution 50 ml of acetone and 7.1 g (50 mmol)of methyliodide was added and the mixture was kept in the dark for 20hours at room temperature. After pouring in 400 ml of diethylether, theformed precipitate was filtered off, washed with diethylether and dried.Yield: 3.25 g (88%).

Nα-(2-naphthylsulfonyl)-3-amidino-(L)-phenylalanine-4methylsulfonylpiperazidehydrochloride (formula I; X=Cl, R² =β-naphthyl, R³ =--SO₂ CH₃)

3.23 g (4.6 mmol) of methyl-thioimidate hydroiodide was dissolved in 110ml of methanol, 0.58 g (7.5 mmol) of ammonium acetate was added to thesolution and the mixture was heated for 3 hours at 60° C. in a waterbath. Afterwards, the solvent was evaporated under reduced pressure, theresidue dissolved in methanol and the amidine hydroiodide precipitatedwith ethyl acetate/diethylether 9:1, filtered off, washed withdiethylether and dried. For the conversion into the hydrochloride, theobtained product was dissolved in methanol and the solution passed overa strongly basic ion exchanger (Amberlite IRA-410, loaded with Cl⁻). Thehydrochloride was precipitated from the concentrated methanolic solutionwith diethylether.

Yield: 2.1 g (79%). [α]_(D) ²⁰ =+70.0° (C=1, in methanol). TLC: R_(f)=0.32 (organic phase of ethyl acetate 4/acetic acid 1/water 2//v/v/v)Specific rotation of the corresponding (D)-configurated compound: []D²⁰=-70.5° (C=1, in methanol)

EXAMPLE 4 Pmc-3-amidino-(L)-phenylalanine-4-methylsulfonylpiperazide

Boc-3-cyano-(L)-phenylalanine

6 g (26.5 mmol) of 3-cyano-(L)-phenylalanine hydrochloride and 9.1 ml(53.2 mmol) of N-ethyldiisopropylamine was suspended in 17 ml of water.To this suspension was added a solution of 7.2 g (29.2 mmol) of2-(Boc-oxyimino)-2-phenylacetonitrile in 20 ml of dioxane and stirringcontinued for 16 hours at room temperature. After subsequent addition of50 ml of water, the solution was extracted with 50 ml of ethyl acetate,the organic phase separated and the pH of the aqueous phase adjusted to3 with dilute hydrochloric acid. After 3 extractions with 100 ml each ofethyl acetate, the collected organic solutions were washed withsaturated sodium chloride solution, dried over magnesium sulfate and thesolvent was evaporated under reduced pressure.

Yield: 6.2 g (81%)

Boc-3-cyano-(L)-phenylalanine-4-methylsulfonylpiperazide

4.92 g (24.5 mmol) of 1-methylsulfonylpiperazine hydrochloride and 2.7ml (24.5 mmol) of NMM was dissolved in 50 ml of DMF. After addition of 4g (29.6 mmol) of HOBt and a solution of 5.93 g (20.4 mmol) ofBoc-3-cyano-(L)-phenylalanine in 200 ml of THF, the mixture was cooledto O° C. 5.1 g (24.7 mmol) of DCC were added and the mixture was stirredfor 48 hours at room temperature. Afterwards, the precipitateddicyclohexyl urea was filtered off, the THF moiety of the solutionevaporated under reduced pressure, filtered and the filtrate poured intoa mixture of 100 ml of 5% sodium bicarbonate solution and 200 ml of icewater. The formed precipitate was filtered off, washed with water,dissolved in methanol and the solvent was evaporated under reducedpressure. To remove the still present water, a co-distillation withtoluene/isopropanol was performed several times. The only slightlyimpure product was processed in this form. Yield: 7.9 g (89%)

3-Cyano-(L)-phenylalanine-4-methylsulfonylpiperazide hydrochloride

7.9 g of the previously described crude product was dissolved in 70 mlof ethyl acetate and 30 ml of diethylether, 50 ml of 2.5 N HCl in ethylacetate was added and the solution was stirred for 48 hours at roomtemperature, whereby the desired hydrochloride crystallized out. Afteraddition of 200 ml of diethylether, the mixture was stirred for 1 hour,the precipitate filtered off, washed with diethylether and dried.

Yield: 4.95 g (73%)

Pmc-3-cyano-(L)-phenylalanine-4-methylsulfonyl piperazide (formula VI;R² =--Pmc, R³ =--SO₂ CH₃)

4.76 g (12.8 mmol) of 3-cyano-(L)-phenylalanine-4-methylsulfonylpiperazide hydrochloride and 1.29 g (12.8 mmol) of NMM was dissolved in25 ml of DMF, 4.64 g (15.3 mmol) of Pmc-chloride and 1.55 g (15.3 mmol)of NMM was added and the mixture was stirred for 48 hours at roomtemperature. Precipitated NMM hydrochloride was then filtered off andthe solvent evaporated under reduced pressure. The residue was taken upin ethyl acetate and the organic phase washed with 0.1 N HCl andsaturated sodium chloride solution. After drying over magnesium sulfate,the solvent was evaporated under reduced pressure. The obtained productwas purified by column chromatography over silica gel 60 with chloroformas the eluent.

Yield: 6.45 g (84%).

[α]_(D) ²⁰ =+10.4° (C=1, methanol) TLC: R_(f) =0.66 (chloroform 40/methanol 4/acetic acid 1//v/v/v)

Pmc-3-thiocarboxamido-(L)-phenylalanine-4-methylsulfonyl piperazide(formula VII;

R² =--Pmc, R³ =--SO₂ CH₃)

6.34 g (0.5 mmol) of the previously described cyano compound wasdissolved in 40 ml of pyridine, 20 drops of TEA were added and thesolution was saturated by introduction of

H₂ S for 10 minutes. The reaction mixture was kept at room temperaturefor 2 days. The solvent was then evaporated under reduced pressure; thesolid residue was suspended in 100 ml of ethyl acetate, briefly heated,filtered off, washed with ethyl acetate and dried.

Yield: 5.86 g (88%).

Pmc-3-S-methyliminothiocarbonyl-(L)-phenylalanine-4-methylsulfonylpiperazide (formula IX; Alk=--CH₃, X=I, R² =--Pmc, R³ =--SO₂ CH₃)

5.86 g (7.52 mmol) of the previously described thioamide was dissolvedunder heating in 11 ml of DMF, 250 ml of acetone and 13 g (92 mmol) ofmethyl iodide was added and the mixture was kept in the dark overnightat room temperature. The mixture was then poured into 1 l ofdiethylether and stirred for 1 hour, whereupon the formed precipitatewas filtered off, washed with diethylether and dried.

Yield: 6.23 g (87%).

Pmc-3-amidino-(L)-phenylalanine-4-methylsulfonyl piperazidehydrochloride (formula I; X=Cl, R² =--Pmc, R³ =--SO₂ CH₃)

6.23 g (8 mmol) of thioimidate hydroiodide was dissolved in 350 ml ofabs. methanol. After addition of 1 g (13 mmol) of ammonium acetate, themixture was heated to 60° C. in a water bath under stirring, whereby aprecipitate formed which needed 4 hours to completely dissolve again.The reaction was followed by thin layer chromatography (TLC) and a totalof 1 g (13 mmol) of ammonium acetate in portions of 0.2, 0.5 and 0.3 gwas added after 2, 4 and 6 hours, respectively. After 8 hours, noinitial compound could be detected anymore by TLC. The solvent was thenevaporated under reduced pressure, the residue dissolved in ethanol andthe amidine hydroiodide precipitated with ethyl acetate/ diethylether1:1. For the conversion into the hydrochloride, the obtained product wasdissolved in methanol and the solution passed over a strongly basic ionexchanger (Amberlite IRA-410, loaded with Cl³¹). The hydrochloride wasprecipitated from the concentrated methanolic solution withdiethylether.

Yield: 3.34 g (64%). [α]_(D) ²⁰ =+47.7° (C=1, in methanol). TLC: R_(f)=0.5 (organic phase of ethyl acetate 4/acetic acid 1/water 2//v/v/v)Specific rotation of the corresponding (D)-configurated compound:[α]_(D) ²⁰ =-48.3° (C=1, in methanol)

All the amidine hydrochlorides were purified by column chromatographyover Sephadex LH-20 with methanol as the eluent.

    ______________________________________                                        Abbreviations                                                                 ______________________________________                                        Ac              acetyl                                                        AcONHiPr        --CH.sub.2 CONHCH(CH.sub.3).sub.2                             BOC             t-butyloxycarbonyl                                            Bzl             benzyl                                                        cBu             cyclobutyl                                                    cHex            cyclohexyl                                                    cPr             cyclopropyl                                                   DCC             dicyclohexylcarbodiimide                                      DMF             dimethylformamide                                             Et              ethyl                                                         EtOH            hydroxyethyl                                                  EtOEtOH         hydroxyethyl-ethoxy                                           For             formyl                                                        HOBt            hydroxybenzotriazole                                          Me              methyl                                                        NMM             N-methylmorpholine                                            Ph              phenyl                                                        TEA             triethylamine                                                 THF             tetrahydrofuran                                               ______________________________________                                    

    ______________________________________                                        LEGENDS TO THE TABLES 1-2                                                     R.sub.1       R.sub.2                                                         ______________________________________                                        AMD = amidino Mtr = 2,3,6-methyl-4-methoxy-phenyl                             AMe = aminomethyl                                                                           TIPP = 2,4,6-triisopropyl-phenyl                                              TMeP = 2,4,6-trimethyl-phenyl                                                 Tol = 4-methyl-phenyl                                           naphthyl      2-Naph = β                                                 naphthyl      1-Naph = α                                                 ##STR7##                                                                      ##STR8##                                                                      ##STR9##                                                                      ##STR10##                                                                    ______________________________________                                    

The biological properties of some representative compounds of thepresent invention are mentioned hereafter:

Table 1 shows the inhibition of the clotting enzyme thrombin as comparedto trypsin by the cited compounds by means of the dissociation constantK_(i) (expressed in μmol/1). All the compounds investigatedcompetitively inhibit the substrate splitting caused by the two enzymes.Among the derivatives of 3-amidinophenylalanine listed in Table 1, thereis a series of compounds having a high antithrombin activity, i.e. withK_(i) -values between 0.1 and 0.001 μmol/1. In most compounds, thethrombin inhibition is more pronounced than the inhibition of trypsin.The K_(i) -values for the inhibition of trypsin are higher by up to oneorder of magnitude than those for thrombin inhibition. A series ofcompounds inhibit thrombin and trypsin with a comparable affinity, whilederivatives, in particular those with defined acyl or heteroarylresidues at the piperazin nitrogen, are potent trypsin inhibitors.

Table 1 also shows the inhibitory activity towards plasmin, factorX_(a), factor XII_(a), plasma kallikrein, glandular kallikrein and tPA.The inhibition of plasmin, factor X_(a) and plasma kallikrein is usuallymuch weaker, the K_(i) -values are higher by 1-2 orders of magnitude.The derivatives are practically ineffective towards factor XII_(a), tPAand glandular kallikrein. Therefore, some compounds are said to beselective thrombin inhibitors, while other derivatives give preferenceto trypsin.

In a series of compounds of the present invention, the toxicity iscomparable to that of the previously investigated derivatives ofbenzamidine-containing amino acids (LD₅₀ 10-50 mg/kg after i.v.application).

The optical antipodes of some derivatives were represented and theirinhibitory effect was studied. According to the results of Turk, D. etal. (FEBS Letters 287, 133-138, 1991), the L-enantiomers were the mosteffective form; as compared to the isomer mixtures, their inhibitoryeffect was increased by a factor of 2. The inhibitory effect of theD-forms is lower by 2 orders of magnitude.

The 2-naphthylsulfonyl protective group can be replaced by an AC--SO₂,Pmc--SO₂, Mtr-SO₂, Cm-SO₂ or TIPP--SO₂ residue. Highly potent inhibitorsare also found.

The compounds that are not characterized with (L) or (D) in Table 1 areracemates and NAPS-F(3AMD)-Pzd(N-COOEt) denotesN-α-(2-naphthylsulfonyl)-(D,L)-3-amidinophenylalanine-4-ethoxycarbonylpiperazide.

                                      TABLE 1                                     __________________________________________________________________________    Inhibition of various trypsin-like serine proteinases by substituted          piperazides of                                                                Nα-protected 3-substituted phenylalanines                               K.sub.i, μmol/l                                                                                                    Factor                                                                             Factor                                                                            Plasma                                                                             Gland.                  R.sup.1                                                                            R.sup.2                                                                           R.sup.3     Nr                                                                              Thrombin                                                                            Trypsin                                                                             Plasmin                                                                            Xa   XIIa                                                                              kallikr.                                                                           kallikr.                                                                          tPA                 __________________________________________________________________________         NAPAP             0.006 0.69  30   7.9  450 14   93  70                  NAPS-F(3AMD)-Pzd(N-COOEt)                                                                          X 0.67  0.022 2.5  54   210 30   >1000                                                                             370                 AMD  2-Naph                                                                            For           0.037 0.22  6.3  19   83  16   440 >1000               AMD(L)                                                                             2-Naph                                                                            For           0.011 0.073 3.3  9.7  42  3.6  >1000                                                                             120                 AMD  2-Naph                                                                            Ac          1 0.023 0.14  8.4  48   210 28   >1000                                                                             >1000               AMD(L)                                                                             2-Naph                                                                            Ac          2 0.012 0.044 5.4  35   150 15   >1000                                                                             440                 AMD  2-Naph                                                                            SO.sub.2 Me   0.0028                                                                              0.19  12   22   86  77   >1000                                                                             >1000               AMD(L)                                                                             2-Naph                                                                            SO.sub.2 Me 5 0.0021                                                                              0.067 9.0  19   52  9.2  >1000                                                                             190                 AMD  2-Naph                                                                            SO.sub.2 Et   0.044 0.19  3.5  24   212 58   >1000                                                                             820                 AMD  2-Naph                                                                            SO.sub.2 -Ph  0.041 0.33  19   24   120 36   >1000                                                                             >1000               AMD  2-Naph                                                                            SO.sub.2 -Tol 0.022 0.22  15   24   >1000                                                                             30   >1000                                                                             >1000               AMD  2-Naph                                                                            SO.sub.2 -2-Naph                                                                            0.024 0.40  11   25   90  22   >1000                                                                             >1000               AMD  2-Naph                                                                            SO.sub.2 -TMeP                                                                              0.075 0.59  31   13   690 1.0  >1000                                                                             >1000               AMD  2-Naph                                                                            SO.sub.2 -Mtr 0.05  1.5   37   25   7.6 6.9  >1000                                                                             >1000               AMD  2-Naph                                                                            CO-N(Me).sub.2                                                                              0.0088                                                                              0.17  6.1  24   200 47   >1000                                                                             >1000               AMD(L)                                                                             2-Naph                                                                            CO-N(Me).sub.2                                                                            6 0.004 0.097 4.9  15   51  7.3  >1000                                                                             >1000               AMD  2-Naph                                                                            CO-N(Me).sub.2                                                                              0.013 0.049 6.2  22   130 28   >1000                                                                             >1000               AMD  2-Naph                                                                            EtOH          0.058 1.1   42   35   >1000                                                                             74   >1000                                                                             >1000               AMD(L)                                                                             2-Naph                                                                            EtOH        3 0.036 0.60  21   22   320 25   >1000                                                                             190                 AMD  1-Naph                                                                            SO.sub.2 Me 11                                                                              0.026 0.32  5.0  9.7  >1000                                                                             79   >1000                                                                             >1000               AMD  1-Naph                                                                            CO.sub.2 N(Me).sub.2                                                                        0.068 0.25  4.8  5.9  72  37   >1000                                                                             270                 AMD  Pmc Ac            0.074 0.60  11.5 150  420 44   760 670                 AMD  Pmc SO.sub.2 Me   0.0074                                                                              0.76  30   75   140 140  >1000                                                                             >1000               AMD(L)                                                                             Pmc SO.sub.2 Me 10                                                                              0.0053                                                                              0.32  10.3 37   >1000                                                                             110  >1000                                                                             600                 AMD  Pmc CO-N(Me).sub.2                                                                              0.033 0.58  13   26   >1000                                                                             93   >1000                                                                             >1000               AMD  Pmc EtOH          0.059 3.3   48   91   >1000                                                                             180  >1000                                                                             >1000               AMD  Mtr SO.sub.2 Me   0.032 1.8   18   120  630 150  >1000                                                                             >1000               AMD(L)                                                                             Mtr SO.sub.2 Me 9 0.020 0.86  8.2  48   >1000                                                                             170  >1000                                                                             600                 AMD(L)                                                                             (-)Cm                                                                             SO.sub.2 Me 7 0.012 0.22  14   1.1  61  25   >1000                                                                             20                  AMD(L)                                                                             (+)Cm                                                                             SO.sub.2 Me 8 0.031 0.44  29   6.7  70  130  >1000                                                                             82                  AMD  TIPP                                                                              Ac            0.098 0.22  3.0  4.0  47  7.3  200 17                  AMD  AC  Ac          4 0.014 0.026 8.6  4.1  52  7.0  >1000                                                                             >1000               AMD  AC  For           0.031 0.033 13   1.7  7.2 11   >1000                                                                             84                  AMD  2-Naph                                                                            2-Pyl         0.52  0.035 3.3  12   84  19   >1000                                                                             >1000               AMD  2-Naph                                                                            2-Pym         1.8   0.061 3.3  14   100 19   >1000                                                                             320                 AMD  2-Naph                                                                            COCH(Me).sub.2                                                                              0.26  0.063 9.3  84   >1000                                                                             50   >1000                                                                             440                 AMD  2-Naph                                                                            COCH.sub.2 OMe                                                                              1.3   0.066 6.2  16   74  16   >1000                                                                             >1000               AMD  2-Naph                                                                            CO(CH.sub.2).sub.4 CH.sub.3                                                                 6.5   0.064 2.6  19   270 23   >1000                                                                             >1000               AMD  2-Naph                                                                            CO-cPr        0.14  0.044 5.1  45   540 22   >1000                                                                             170                 AMD  2-Naph                                                                            CO-cBu        2.5   0.026 7.6  58   >1000                                                                             56   >1000                                                                             >1000               AMD  2-Naph                                                                            CO-cHex       36    0.072 10.2 58   350 79   >1000                                                                             >1000               AMD  2-Naph                                                                             ##STR11##    2.5   0.081 2.7  16   72  19   >1000                                                                             170                 Ame  2-Naph                                                                            2-Pyl         24    0.53  26   130  >1000                                                                             94   >1000                                                                             >1000               __________________________________________________________________________

Table 2 is an overview of all the synthesized and in vitro-testedcompounds which are not included in Table 1.

                                      TABLE 2                                     __________________________________________________________________________    Summary of the synthesized compounds of general formula I, not mentioned      in Table 1                                                                    R.sup.1                                                                            R.sup.2                                                                           R.sup.3    R.sup.1                                                                            R.sup.2                                                                           R.sup.3  R.sup.1                                                                          R.sup.2                                                                           R.sup.3                          __________________________________________________________________________    AMD(D)                                                                             2-Naph                                                                            Ac         AMD  2-Naph                                                                            SO.sub.2 CF.sub.3                                                                      AMe                                                                              2-Naph                                                                            SO.sub.2 Et                      AMD(D)                                                                             2-Naph                                                                            EtOH       AMD  2-Naph                                                                            SO.sub.2 N(Me).sub.2                                                                   AMe                                                                              2-Naph                                                                            SO.sub.2 CF.sub.3                AMD(D)                                                                             2-Naph                                                                            For        AMD  2-Naph                                                                            SO.sub.2 -1-Naph                                                                       AMe                                                                              2-Naph                                                                            SO.sub.2 N(Me)                   AMD  Cm  Ac         AMD  2-Naph                                                                            TIPP     AMe                                                                              2-Naph                                                                            SO.sub.2 -Ph                     AMD(L)                                                                             Tol Ac         AMD  2-Naph                                                                            SO.sub.2 -Pmc                                                                          AMe                                                                              2-Naph                                                                            SO.sub.2 -Tol                    AMD  2-Naph                                                                            AcONHiPr   AMD  2-Naph                                                                            SO.sub.2 -(-)Cm                                                                        AMe                                                                              2-Naph                                                                            TIPP                             AMD  2-Naph                                                                            SO.sub.2 CH(Me).sub.2                                                                    AMD  2-Naph                                                                            Bzl      AMe                                                                              2-Naph                                                                            SO.sub.2 -2-Naph                 AMD  2-Naph                                                                            EtOEtOH    AMD  Tol Ac       AMe                                                                              2-Naph                                                                            2-Pym                            AMD  2-Naph                                                                            H          AMD  TIPP                                                                              EtOH     AMe                                                                              Tol Ac                               AMD  2-Naph                                                                            CH.sub.2 CO--N                                                                           AMD  TIPP                                                                              CON(Me).sub.2                                                                          AMe                                                                              Mtr Ac                                                   AMD  TIPP                                                                              SO.sub.2 Me                                                                            AMe                                                                              Mtr SO.sub.2 Me                      AMD  2-Naph                                                                            COEt       AMD  TIPP                                                                              SO.sub.2 -2-Naph                                                                       AMe                                                                              Pmc Ac                               AMD  2-Naph                                                                            COCH.sub.2 CH(Ph).sub.2                                                                  AMD  Mtr Ac       AMe                                                                              Pmc EtOH                             AMD  2-Naph                                                                            COC(Me).sub.3                                                                            AMD  Mtr EtOH     AMe                                                                              Pmc SO.sub.2 Me                      AMD  2-Naph                                                                            CO-Ph      AMD  Mtr CON(Me).sub.2                                                                          AMe                                                                              Pmc 2-Pyl                            AMD  2-Naph                                                                            CO-Ph-OMe(p)                                                                             AMD  Mtr SO.sub.2 -2-Naph                                                                       AMe                                                                              AC  Ac                               AMD  2-Naph                                                                            CO-(2)-Naph                                                                              AMD  1-Naph                                                                            Ac       AMe                                                                              2-Naph                                                                            For                              AMD  2-Naph                                                                            CO-CH.sub.2 -NH.sub.2                                                                    AMD  1-Naph                                                                            EtOH     AMe                                                                              2-Naph                                                                            Ac                               AMD  2-Naph                                                                            CO-CH.sub.2 NHCOCOMe                                                                     AMD(D)                                                                             Pmc SO.sub.2 Me                                                                            AMe                                                                              2-Naph                                                                            H                                AMD  2-Naph                                                                            CO-CH.sub.2 NHBoc                                                                        AMD  Pmc SO.sub.2 -2-Naph                                                                       AMe                                                                              2-Naph                                                                            COEt                             AMD  2-Naph                                                                            COCOOME    AMD  Pmc 2-Pyl    AMe                                                                              2-Naph                                                                            CON(Me).sub.2                    AMD  2-Naph                                                                            CH.sub.2 COOMe                                                                           AMD  Cm  Ac       AMe                                                                              2-Naph                                                                            SO.sub.2 Me                      AMD(D)                                                                             2-Naph                                                                            CO-N(Me).sub.2                                                                           AMD  2-Naph                                                                             ##STR12##                                                                             AMe                                                                              2-Naph                                                                             ##STR13##                       AMD  2-Naph                                                                            CO-N(Et).sub.2                                                                           AMD  2-Naph                                                                             ##STR14##                                       AMD(D)                                                                             2-Naph                                                                            SO.sub.2 Me                                                          __________________________________________________________________________

Tables 3-5 show the results of studies on the pharmacokinetics ofrepresentative compounds of the present invention and, for comparison,the values with NAPAP. Table 6 shows the results obtained with selectedcompounds after intraduodenal application. The compounds to be testedwere administered to rats intravenously (Table 3), perorally (Table 4),rectally (Table 5) and intraduodenally (Table 6), respectively. Afteradministration, blood samples were taken from the test animals at timeintervals from 2 to maximally 360 minutes and the blood level of thecompounds to be tested was determined by HPLC.

                                      TABLE 3                                     __________________________________________________________________________    Concentration (ng/ml) of selected compounds in the plasma                     of rats after intravenous administration of 1 mg/kg                           Time                                                                              Compound                                                                  (min)                                                                             NAPAP                                                                              No. X                                                                              No. 1                                                                              No. 2                                                                              No. 3                                                                              No. 5                                                                              No. 6                                       __________________________________________________________________________     5  1150 812  1058 780  2210 879  823                                         15  428  169  394  224  145  367  289                                         30  376  40   150  95   313  151  194                                         60  192  8    89   38   112   50  141                                         120  84  0    74   12    0    0    86                                         180 106  0    51   10    0    0    70                                         __________________________________________________________________________

                  TABLE 4                                                         ______________________________________                                        Concentration (ng/ml) of selected compounds in the plasma                     of rats after oral administration of 50 mg/kg                                 Time     Compound                                                             (min)    NAPAP    No. X       No. 1 No. 2                                     ______________________________________                                         30      0        33          87    1197                                       60      0        10          63    873                                       120      0        0           57    333                                       180      0        0           69    124                                       240      0        0           91     44                                       300      0        0           61     46                                       ______________________________________                                    

                                      TABLE 5                                     __________________________________________________________________________    Concentration (ng/ml) of selected compounds in the plasma                     of rats after rectal administration                                                   Compound                                                              Time                                                                              Dose                                                                              NAPAP                                                                              No. X                                                                             No. 1                                                                              No. 2                                                                             No. 3                                                                             No. 5*                                                                             No. 6                                      (min)                                                                             mg/kg                                                                             100  100 100  100 20  20   20                                         __________________________________________________________________________     30     18   3255                                                                              31406                                                                              32448                                                                             7260                                                                              1650 1179                                        60     0    2371                                                                              17862                                                                              23132                                                                             3800                                                                              958  449                                        120     0    1804                                                                              3776 5302                                                                              1740                                                                              299  850                                        180     0    898 1075 2696                                                                               814                                                                              192  272                                        240     0    626 438  1263                                                                               215      46                                        300     0    592 220   810                                                                               61                                                 360     0    448 158   508                                                                               34                                                 __________________________________________________________________________     *free base of the compound                                               

                  TABLE 6                                                         ______________________________________                                        Concentration (ng/ml) of selected compounds in the plasma                     of rats after intraduodenal administration of 100 mg/kg                       Time  Compound                                                                (min) No. 2   No. 3    No. 3*                                                                              No. 5  No. 5*                                                                              No. 6                               ______________________________________                                         30   21691   3824     2262  8744   1576  20460                                60   15152   2172     795   9314   1900  6495                                120   4275    1457     361   8612   1763  3284                                180   2598     945     228          297   2064                                240   2185    1277     144          530   1500                                300   2195    1400     276          137   1203                                360   2847                          119   1437                                ______________________________________                                         *free base of the compound                                               

The derivatives investigated show an improved pharmacokinetic behaviournot only in comparison to NAPAP, but also to the previously describedpiperazide X. Although compounds 1, 2, 3, 5 and 6 of the presentinvention are eliminated at comparable rate after intravenousadministration and only slightly resorbed after oral application, inpart very high blood levels lasting for 1 to 2 hours are found afterrectal administration. After rectal administration, NAPAP cannot bedetected in plasma, while some of the representative compounds tested inthe present invention reach extremely high concentrations. Compounds 1,2 and 3 can be detected in plasma even after 6 hours. The plasma levelsobtained after rectal administration are considerably higher than thoseof the previously described piperazide X. Also the plasma concentrationsmeasured after intraduodenal application, partly after several hours,are considerable.

In vitro, some of the representative compounds of the present inventionhave an extraordinary anticoagulant activity. In all cases, the thrombintime (TT) was the most prolonged value. This corresponds to theselectivity of these inhibitors which, among the clotting factors,inhibit thrombin most effectively. This is illustrated for compounds 1to 11 in Table 7. Prolongation of the activated partial thromboplastintime (aPTT), which is also influenced, besides thrombin, by the enzymeswhich participate in the early phase of coagulation, is obtained byhigher inhibitor concentrations. This also applies to the influence ofthe prothrombin time (PT) which represents the extrinsic coagulationpathway. Table 7 shows the concentrations that are necessary to doublethe clotting times. For the effective thrombin inhibitors 1, 2, 4, 5, 6and 7, the value amounts to less than 10⁻⁷ mol/1 for the prolongation ofthe TT, to 1 μmol/1 for the prolongation of the aPTT and PT. The activeinhibitors NAPAP and compound X tested for comparison purposes areefficient according to their K_(i) -value.

In plasma, inhibitors of the piperazine type are (absolutely) stable.Incubation in human plasma at 37° C. did not lead to any change in theinhibitory activity for 5 hours.

                  TABLE 7                                                         ______________________________________                                        Inhibition of coagulation in human plasma                                     by selected compounds                                                         Thrombin     Concentr. [μmol/l] to double the                                     inhibition                                                                              Thrombin                                                     No.    K.sub.i, μmol/l                                                                      time       aPTT  Prothrombin time                            ______________________________________                                        NAPAP  0.006     0.048      0.50  1.0                                         X      0.67      4.1        20    45                                          1      0.023     0.095      0.90  2.5                                         2      0.012     0.055      0.36  0.90                                        3      0.036     0.14       0.65  1.3                                         4      0.014     0.085      1.2   2.0                                         5      0.0021    0.034      0.26  0.39                                        6      0.004     0.042      0.3   0.65                                        7      0.012     0.075      0.55  1.0                                         8      0.031     0.13       1.2   2.0                                         9      0.020     0.12       0.57  1.1                                         10     0.0053    0.10       0.44  0.8                                         11     0.026     0.22       1.8   3.1                                         ______________________________________                                    

The anticoagulant effect of the compounds can also be demonstrated invivo. After rectal administration of the compounds to be tested, theanticoagulant effect was determined in the plasma of experimentalanimals. This is illustrated for compounds 2, 3, 5 and 6 in Table 8.Like the concentration progression determined by means of HPLC inplasma, the antithrombin effect can be detected in the clotting test.

                  TABLE 8                                                         ______________________________________                                        Inhibition of coagulation in rat plasma after rectal                          application of compounds 2, 3, 5 and 6                                                Concentration                                                         Time    in plasma     Clotting time (sec)                                     (min)   (ng/ml)       Thrombin time                                                                             aPTT                                        ______________________________________                                        Compound 2  20 mg/kg                                                           0      0             64          28                                           30     5440          >300        120                                          60     2090          >300        87                                          120     812           >300        59                                          180     660           >300        50                                          Compound 2  5 mg/kg                                                            0      0             32          22.5                                         30     296           >300        37                                           60     160           224         33                                          120     60            176         30                                          180     40            107         26.4                                        Compound 3  5 mg/kg                                                            0      0             45          23.5                                         30     897           300         39.8                                         60     462           298         32.8                                        120     355           165         27.4                                        180     49            114         26.0                                        Compound 5  20 mg/kg                                                           0      0             153         22                                           30     1572          >300        65                                           60     983           >300        62.5                                        120     380           >300        36                                          180     195           >300        29.7                                        Compound 5  5 mg/kg                                                            0      0             51.5        21.2                                         30     168           >300        34.5                                         60     65            255         30.5                                        120     82            137.5       27.5                                        180     10            95          25.3                                        Compound 6  5 mg/kg                                                            0      0             55.3        22.3                                         30     648           >300        34.5                                         60     362           >300        31.5                                        120     157           >300        28.4                                        180     83            213         26                                          ______________________________________                                    

After rectal application, the compounds of the present invention areabsorbed to an extent leading to antithrombotically active plasmalevels. This has been shown with the model of the stasis-induced venousthrombosis in the rat according to Wessler et al. This method consistedin macroscopically evaluating thrombi induced by serum in jugular veinsegments by means of a scale (0=liquid blood, 1=one or several smallthrombi, 2=not fully closed vascular segment, 3=fully closed vascularsegment). The antithrombotic effect is clearly dose-dependent. At a doseof 100 or 20 mg/kg, respectively, the thrombus formation is completelyinhibited; at the lower dose of 5 mg/kg, vascular occlusion is largelyprevented or thrombus formation is greatly enhanced, respectively.

                  TABLE 9                                                         ______________________________________                                        Dose-dependent antithrombotic efficacy after rectal                           application of compound 2 on the model of                                     stasis-induced venous thrombosis in the rat                                                Plasma level Thrombus  relative                                  Dose         (ng/ml)      scale     thromobus                                 (mg/kg)                                                                              n     30 min  60 min 0   1   2   3   scale                             ______________________________________                                        0      5     0       0      0   0   1   9   1                                 100    3     28698   16118  6   0   0   0   0                                 20     4     1617    998    6   1   0   0   0.05                              5      5     228     160    0   8   2   0   0.41                              ______________________________________                                         n = number of experimental animals each having generally 2 vein segments      evaluated.                                                               

The relative thrombus scale is the quotient of the mean thrombus size ofthe treated group (e.g. the mean value at 5 mg/kg is 1.2) and the meanthrombus size of the control group (2.9).

The piperazides synthesized according to the method described in thepresent invention and used as such or as salts with a physiologicallycompatible mineral or organic acid can be converted in appropriate formsof application by applying adequate auxiliaries and carriers.Corresponding to the pharmacological behaviour, tablets, drag ees,suppositories and solutions are particularly significant.

The dosage depends above all on the antithrombin activity, theattainable blood level with the corresponding form of application, thebioavailability as well as the patient's general constitution, whereby asufficient antithrombotic activity can be reached with dosages between0.5 and 50 mg/kg.

By means ofNα-(2-naphthylsulfonyl)-(L)-3-amidinophenylalanine-4-acetylpiperazidehydrochloride (compound 2), the conversion into 3 pharmaceutical formsof administration should be representatively shown.

EXAMPLE 1

Tablets with 20 mg of compound 2 as the active substance, coated with agastric juice-resistant protective lacquer

Composition: Core: 20 mg of active substance, 96 mg of lactose, 3 mg oftalcum and 1 mg of magnesium stearate. Coating: 23.92 mg of Eudragit S12.5 P (R ohm Pharma, Darmstadt), 0.266 mg of dibutylphthalate, 0.744 mgof talcum, 23.92 mg of acetone/ethanol 1+1.

Manufacturing process: The active substance mixed with the additives ispressed through a 0.5 mm-meshed sieve and, once dried, formed into ovaltablet cores. The protective lacquer is then sprayed in a specialgranulator, whereupon the lacquered cores are dried.

EXAMPLE 2

Suppositories with 10 mg of compound 2 as the active substance.

Composition: 1 suppository contains 10 mg of active substance and 1 g ofWitepsol W45 as the basic substance.

Specifications for the manufacture of 10 suppositories: 100 mg of thefinely powdered active substance are ground with the liquefied basicsubstance. The preparation is mixed portionwise with the remainingliquefied basic substance and worked until a regular quality isobtained. Nearly at the limit of pourability, the mixture is poured inan adequate form and allowed to cool down at rest.

EXAMPLE 3

Injection and infusion solution, respectively, with 2.5 mg/ml ofcompound 2 as the active substance.

Manufacturing process: 0.25 g of active substance is diluted in 100 mlof water for injection, whereafter the solution is filtered and, ifnecessary, filled into 2 ml ampoules. The closed containers filled withthis solution (infusion bottles, ampoules) are submitted to a steamsterilization at 121° to 124° C.

We claim:
 1. D,L-, L- or D-phenylalanine piperazides of formula##STR15## wherein R¹ represents a basic group of formula ##STR16## R²represents an (un)substituted aryl or heteroaryl residue, and R³represents an acyl residue of formula --COX, wherein X=H, unbranched orbranched, possibly substituted alkyl, or (un)substituted aryl orcycloalkyl,an aralkyl residue in which the aromatic residue may besubstituted, a carboxamide residue of formula --CONR'R", athiocarboxamide residue of formula --CSNR'R" or an ethylamide residue offormula --CH₂ --CONR'R" in which R'=R"=H; R'=R"=alkyl; R'=H, R"=alkyl;R'=H, R"=aryl, or R' and R" may form a cycloaliphatic orheterocycloaliphatic ring with the nitrogen atom, an SO₂ --Y residue inwhich Y means (un)substituted alkyl, (un)substituted aryl or heteroarylor --NR'R", in which R' and R"=H may be equal or not to low alkylC_(1-C) ₃, a cycloaliphatic ring with 5 to 8 C atoms which may besubstituted with a hydroxyl or oxo group, an (un)substituted heteroarylresidue or a heterocycloaliphatic residue, respectively, afunctionalized alkyl residue of formula --(CH₂)_(n) --X wherein thealkyl chain may be unbranched or branched, n=1 to 8 and the functionalresidue X representsa hydroxyl group the H atom of which can besubstituted, a halogen atom, a tertiary amino group of formula --N(Alk)₂wherein the alkyl groups have 1 to 3 C atoms, and the alkyl groups aresame, and the nitrogen atom may belong to a cycloaliphatic ring with 5to 7 ring parts to which one or two further rings may be added, anacylaminomalonate group of formula ##STR17## an ##STR18## group or a##STR19## group, and the salts thereof with mineral or organic acids. 2.Phenylalanine piperazides according to claim 1, wherein the(un)substituted aryl or heteroaryl residue in R² represents phenyl,4-methylphenyl, 2,4,6-trimethyl- or -triisopropylphenyl,4-methoxy-2,3,6-trimethylphenyl, 2,2-dimethyl-6-methoxy- or2,2,5,7,8-pentamethylchromanyl, anthraquinonyl, 1- or 2-naphthyl,quinolyl- or isoquinolyl, or a camphor residue, respectively. 3.Phenylalanine piperazides according to claim 1, wherein X in the acylresidue in R³ is lower alkyl or cycloalkyl.
 4. Phenylalanine piperazidesaccording to claim 1, wherein the aromatic residue of the aralkylresidue in R³ is substituted with a halogen atom, an alkyl, alkoxy,hydroxy or nitro group.
 5. Phenylalanine piperazides according to claim1, wherein the alkyl residue in the SO₂ --Y residue is methyl,trifluoromethyl or trichloromethyl.
 6. Phenylalanine piperazidesaccording to claim 1, wherein the aryl or heteroaryl residue in the SO₂--Y residue is phenyl, 4-methylphenyl, 2,4,6-trimethyl- or-triisopropylphenyl, 4-methoxy-2,3,6-trimethylphenyl,2,2-dimethyl-6-methoxy- or 2,2,5,7,8-pentamethylchromanyl,anthraquinonyl, naphthyl or quinolyl, or O-aryl, respectively. 7.Phenylalanine piperazides according to claim 1, wherein the(un)substituted heteroaryl residue in R³ is pyridyl or pyrimidyl, or theheterocycloaliphatic residue in R³ is N-methylpiperidyl, respectively.8. Phenylalanine piperazides according to claim 1, wherein the H atom ofthe hydroxyl group in the functionalized alkyl residue in R³ issubstituted with an alkyl, aralkyl, aryl, hydroxyalkyl or acyl group. 9.Phenylalanine piperazides according to claims 1, wherein Ac in theacylaminomalonate group of formula AcHN--C(COOAlk)₂ and in theAcHN--CH--COOH group of the functionalized alkyl residue means formyl oracetyl and Alk=low alkyl C₁ --C₃.
 10. Phenylalanine piperazidesaccording to claim 1, wherein R¹ represents a basic group of formula(a)=amidino, R² means a β-naphthyl, anthraquinone,2,4,6-triisopropylphenyl or 2,2,5,7,8-pentamethylchroman group, and R³represents an acyl residue, a functionalized alkyl residue, an SO₂ --Yresidue, a carboxyamide residue or heteroaryl residue.
 11. Thephenylalanine piperazides according to claim 3, wherein X in the acylresidue in R³ is methyl or C₃ to C₁₀ cycloalkyl.
 12. The phenylalaninepiperazides according to claim 10, wherein R³ is formyl, acetyl,2-hydroxyethyl, 2-pyridyl, or 2-pyrimidyl.
 13. Method for the synthesisof phenylalanine piperazides according to claim 1, which comprisesconverting a (D,L)-3-cyanophenylalanine alkylester of formula II##STR20## with a sulfochloride R² --SO₂ Cl, wherein R² has thedenotation given in claim 1 or 2, into a racemic compound of formula IV##STR21## from which racemates of the sulfonylated cyanophenylalaninesof formula V ##STR22## are obtained by hydrolysis, or converting thecompounds of formula IV by enzymatic ester hydrolysis with chymotrypsininto the L-configurated amino acids of formula V and converting theD-configurated amino alkyl carboxylates of formula IV obtained by theenzymatic ester hydrolysis into the D-configurated amino carboxylicacids of formula V by hydrolysis, converting the compounds of formula Vby coupling with a piperazine derivative of formula VII ##STR23## intothe (D,L)-, D- or L-cyano compounds with piperazide structure of formulaVI ##STR24## synthesizing the thioamide of formula VIII ##STR25## byaddition of H₂ S to the cyano function, synthesizing a thioimidatehalide of formula IX ##STR26## by conversion with an alkyl halide, orsynthesizing the imidate halides of formula X ##STR27## from the cyanocompounds with piperazide structure of formula VI, and converting thecompound of formula IX with ammonium acetate or the compound of formulaX in an alcoholic ammonia solution into compounds of formula XI##STR28## wherein X means halogen, preferentially chlorine.
 14. Themethod of claim 13, further comprising converting the compounds offormula IX with ammonium acetate into compounds of formula XI, wherein Xis chloride.
 15. A method of achieving an anticoagulant or anti-thrombininduced coagulation effect in a subject, comprising, administering to asubject a pharmaceutically effective dosage of at least one of thephenylalanine piperazides according to claim 1 in the form of apharmaceutically acceptable salt or free base, thereby producing ananticoagulant or anti-thrombin induced coagulation effect in thesubject.
 16. The method of claim 15, further comprising administering atleast one phenylalanine piperazide in the form of an antithromboticallyactive drug subcutaneously, intravenously, orally, rectally, orintraduodenally.
 17. Antithrombotically active drug to be administeredsubcutaneously or intravenously, orally, rectally, or duodenally, inwhich an efficient quantity of at least one phenylalanine piperazideaccording to claim 1 and appropriate additives are present. 18.Antithrombotically active drug according to claim 17, in the form oftablets, drag ees, capsules, pellets, suppositories, solutions,injections or transdermal systems.
 19. Method for blood coagulation orthrombin or trypsin inhibition, in living organisms, characterized bythe administration of an effective quantity of a phenylalaninepiperazide according to claim 1 or of an antithrombotically active drugaccording to claim 17 or 18.